1. Field of the Invention
This invention relates to hybridoma cell lines and monoclonal antibodies produced therefrom which may be used to detect hygromycin B, particularly in animal tissues and feeds.
2. Description of the Prior Art
The anthelmintic, hygromycin B (HB), is an FDA approved feed additive used to control roundworms, nodular worms, and whipworms in swine, and large roundworms, cecal worms, and capillary worms in poultry. It is often used in conjunction with other drugs to increase the rate of weight gain and feed efficiency. Although approved as a feed additive, a zero tolerance level for hygromycin B in swine and poultry products (i.e. tissues, eggs) has been set by the FDA (21 CFR 556.330). For enforcement purposes, a residue limit of 1.4 ppm of the parent compound in the kidney has been established, and these limits are used by the U.S. Department of Agriculture/Food Safety Inspection Service (USDA/FSIS) to detect adulterated products. In order to comply with the regulations, medicated feed must be withdrawn 15 days prior to slaughter in swine and 3 days prior to slaughter in poultry.
Conventional methods for detection of hygromycin B in tissues include a microbiological assay (Johnston et al., J. Food Prot., 1981, 44:828-831) and a high-performance liquid chromatography (HPLC) method (McLaughlin and Henion, J. Chromatog., 1992, 59:195-206). The microbiological assay is nonspecific and requires long incubation periods. The HPLC method is labor intensive, has a multistep sample cleanup, and requires expensive equipment. A polyclonal-based radioimmunoassay (RIA) to detect hygromycin B in feed has been described (Foglesong and LeFeber, J. Assoc. Off. Anal. Chem, 1982, 65:48-51). This RIA overcomes some of the disadvantages of the previous methods, but it suffers from the problems associated with the need to continually synthesize unstable radiotracers and dispose of radiological waste. The disadvantages associated with these assay methods have prevented their being used for routine screening of large numbers of samples.
Enzyme-linked immunosorbent assays have been successfully developed as alternatives to the conventional microbiological or chemical methods for detection of pesticides (insecticides and herbicides), drug residues, and undesirable natural products (Azcona-Olivera et al., J. Agric. Food Chem, 1992, 40:531-534; Candlish et al., J. Assoc. Off. Anal. Chem., 1988, 71:96-964; Degand et al., J. Agric. Food Chem, 1992, 40:70-75; Groopman et al., Proc. Natl. Acad. Sci, 1984, 81:7728-7731; Hu et al., J. Food Prot., 1984, 47:126-127; Jung et al., J. Agric. Food Chem., 1989, 37:1183-1187; Plhak and Sporns, J. Agric. Food Chem., 1992, 40:2533-2540; Roseman et al., J. Agric. Food Chem., 1992, 40:1008-1014; Shelby et al., J. Agric. Food Chem., 1992, 40:1090-1092; Wong and Ahmed, J. Agric. Food Chem., 1992: 40:811-816; Woychik et al., Appl. Env. Microbiol., 1984, 48:1096-1099; Xu et al., J. Assoc. Off. Anal. Chem., 1988, 71:945-948). In contrast to microbiological assays, immunoassays are highly specific (Stanker et al., Toxicology, 1987, 45:229-243; Vanderlaan et al., Carcinogenesis, 1988, 9:153-160; Van Emon et al., Anal. Chem., 1985, 58:1866-1873), and unlike conventional chemical assays, they require minimal sample preparation procedures. However, an efficacious immunoassay requires high levels of specificity and sensitivity to the target agent.
In developing antibodies for use in feed contaminant immunoassays, various strategies have been employed in order to increase the probability that the antibodies will have high-affinity for the hapten. These strategies include (i) maintaining the structure of potentially important determinant groups on the hapten (Sheth and Sporns, J. Agric. Food Chem., 1991, 39:1696-1700); (ii) using or extending the length of a linker arm (McAdam et al., J. Agric. Food Chem., 1992, 40:1466-1470); and (iii) varying the immunization protocol (ie. mode of injection, number of injections, or length of immunization schedule) (Vallejo et al., J. Agric. Food Chem., 1982, 30:572-580). Others also have reported effects of the linker arm length on the specificity and the sensitivity of the resulting antibodies (Wie and Hammock, J. Agric. Food Chem., 1984, 32:1294-1301; Vallejo et al., (ibid.), Harrison et al., J. Agric. Food Chem., 1991, 39:122-128; Sheth and Sporns, (ibid.). In general, for small haptens, extending the hapten away from the protein mass has been helpful or even critical to the production of high-affinity antibodies (Hastings et al., J. Agric. Food Chem., 1988, 36:404-408; Paxton et al., J. Immunol. Meth., 1976, 10:317-327). The factors that exactly control generation of a strong immune response, particularly to hygromycin B, are not clearly understood.